Analysis of P210bcr-abl tyrosine protein kinase activity in various subtypes of Philadelphia chromosome-positive cells from chronic myelogenous leukemia patients

An altered c-abl gene product (P210bcr-abl) possessing associated tyrosine protein kinase activity was recently been reported in several blast chronic myelogenous leukemia (CML) cell lines. We have examined different morphological types of leukocytes directly obtained from patients at the blast crisis stage of CML for expression of P210bcr-abl tyrosine protein kinase activity. Phosphorylation of P210bcr-abl in an immune complex kinase assay using an anti-v-abl peptide serum was observed in blast cells from four Philadelphia chromosome (Ph1)-positive CML patients in blast crisis. P210bcr-abl protein kinase activity was detected regardless of whether the blast cells were of myeloid, lymphoid, or undifferentiated morphology. P210bcr-abl protein kinase activity was not detected in immune complexes either from leukocytes of four Ph1-negative CML patients in blast crisis, of five acute myelogenous leukemia patients, or in the promyelocytic cell line HL-60. Mature myeloid cells are associated with an inhibitory factor for not only P210bcr-abl protein kinase activity, but also protein kinases in general. Therefore, analyses of Ph1-positive benign phase CML myeloid cells, the majority of which are well differentiated, could not be successfully performed. The inhibition of P210bcr-abl protein kinase activity is not a specific property of mature cells from CML patients since granulocytes from a normal volunteer also demonstrated a similar effect. However, extracts of Ph1-positive cultured B-lymphocytes from a patient in benign phase demonstrated active P210bcr-abl protein indicating that the P210bcr-abl protein is expressed in an enzymatically active form in the earlier phases of CML. In addition to the previously reported P210 and P190 abl-related proteins, a novel Mr 53,000 protein was found to undergo phosphorylation at serine and tyrosine in immune complex kinase assays of two blast crisis CML cell lines (K562 and EM2) and in samples from blast crisis patients in which P210bcr-abl was detected. Peptide mapping by the Cleveland technique suggested that Mr 53,000 protein is unrelated to P210bcr-abl. Immune complex kinase assays of K562 cells with an anti-src serum (GD-11) yielded active c-src kinase and a Mr 50,000 phosphorylated protein, both of which were resistant to alkaline hydrolysis. Peptide mapping suggested that Mr 53,000 protein is related to Mr 50,000 protein which is precipitated with P210bcr-abl as an Mr 300,000 protein complex.     

Novel targeted therapies for Bcr-Abl positive acute leukemias: beyond STI571

In the pathophysiology of CML, the constitutive activity of the Bcr-Abl tyrosine kinase (TK) is, most likely, the sole molecular abnormality of the chronic phase. It also remains a critical molecular determinant of malignant behavior of the leukemic progenitors in the accelerated and blastic phase of CML. Therefore, downregulation of the levels and activity of Bcr-Abl is clearly the lynchpin of a rational therapeutic strategy against all phases of CML. Support for this has only been strengthened by the observations that resistance to imatinib mesylate (imatinib) commonly involves a breakthrough and the persistent activity of Bcr-Abl TK. This is due to either mutations that inhibit imatinib action on Bcr-Abl TK or amplification of the bcr-abl gene. Recent studies have demonstrated that other small molecule tyrosine kinase inhibitors that also inhibit Bcr-Abl TK may be highly active in inducing differentiation and apoptosis of CML progenitors, regardless of their sensitivity to imatinib. Small molecule inhibitors that downregulate the levels of Bcr-Abl by inhibiting its translation, e.g., arsenic trioxide, or promoting its proteasomal degradation, e.g., geldanamycin analogues, have also been identified. Finally the identification of other potent survival and antiapoptotic signaling pathways in imatinib-resistant CML progenitors indicates that inhibitors of these pathways will eventually be treatment strategies for advanced phases of CML.     

Stem cell factor and chronic myeloid leukemia CD34+ cells

Normal hematopoiesis is a tightly regulated process involving a balance between signals that stimulate and those that inhibit the proliferation and differentiation of hematopoietic progenitors. In chronic myeloid leukemia (CML) there is a perturbation of these controlling elements, resulting in overgrowth of leukemic cells in the bone marrow and spleen. In part, the proliferation of CML CD34+ cells may result from an abnormal response to the cytokine Stem Cell Factor (SCF). SCF induced proliferation and adhesion to the extracellular matrix via fibronectin are not coupled in CML as they are in normal cells and this may contribute to the accumulation of leukemic progenitors. We have previously shown that CD34+ CML cells and the more primitive CD34+ CD38- CML cells do not require the addition of synergistic cytokines to cultures, but are capable of proliferation in SCF alone, and that leukemic CFU-GM are selectively supported in these cultures. In the presence of other cytokines the response of CML cells to SCF is no greater than that of cells from normal donors, suggesting that the leukemic cells are not more sensitive to SCF, but that accessory pathways are already activated in these cells. Cells from patients with myeloproliferative disorders show variable proliferative response to SCF as the sole mitogenic stimulus, suggesting that expression of bcr-abl is essential for proliferation in this cytokine. Further studies to identify the key determinants of the abnormal response to SCF in CML may lead to a better understanding of the proliferative abnormality that underlies CML.     

Chronic Myeloid Leukemia Stem Cell Biology

Leukemia progression and relapse is fueled by leukemia stem cells (LSC) that are resistant to current treatments. In the progression of chronic myeloid leukemia (CML), blast crisis progenitors are capable of adopting more primitive but deregulated stem cell features with acquired resistance to targeted therapies. This in turn promotes LSC behavior characterized by aberrant self-renewal, differentiation, and survival capacity. Multiple reports suggest that cell cycle alterations, activation of critical signaling pathways, aberrant microenvironmental cues from the hematopoietic niche, and aberrant epigenetic events and deregulation of RNA processing may facilitate the enhanced survival and malignant transformation of CML progenitors. Here we review the molecular evolution of CML LSC that promotes CML progression and relapse. Recent advances in these areas have identified novel targets that represent important avenues for future therapeutic approaches aimed at selectively eradicating the LSC population while sparing normal hematopoietic progenitors in patients suffering from chronic myeloid malignancies.     

Circulating myeloid dendritic cell directly isolated from patients with chronic myelogenous leukemia are functional and carry the bcr-abl translocation

Leukemic bcr-abl positive dendritic cells (DCs) are likely to be present in vivo in chronic myelogenous leukemia (CML) patients, but no data are available on their functional qualities. We analyzed the circulating BDCA-1+ myeloid DC compartment in 15 chronic phase CML patients. Phenotypic features of CML DCs were comparable with that of normal DCs, except for the CD80 and CD40 antigens, significantly under-represented in CML patients. Nonetheless, no differences were found between normal samples and leukemic DCs in the allostimulatory ability, as well as in the production of cytokines and polarization of T cell responses. CML DCs were analyzed by fluorescence in situ hybridization (FISH) and found positive for the bcr-abl translocation. However, when bcr-abl+ DCs were tested for their ability to stimulate an autologous T-cell response in vitro, we could not detect a specific recognition. We conclude that an apparently normal circulating DC compartment carrying the Ph+ chromosome can be identified in CML patients; however, these cells appear unable to trigger a protective anti-leukemic immune response in autologous T cells.     

Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease.

Xenograft models of chronic phase human chronic myeloid leukemia (CML) have been difficult to develop because of the persistence of normal hematopoietic stem cells in most chronic phase CML patients and the lack of methods to selectively isolate the rarer CML stem cells. To circumvent this problem, we first identified nine patients' samples in which the long-term culture-initiating cells were predominantly leukemic and then transplanted cells from these samples into sublethally irradiated NOD/SCID and NOD/SCID-beta2microglobulin-/- mice. This resulted in the consistent and durable (>5 months) repopulation of both host genotypes with similar numbers of BCR-ABL+/Ph+ cells. The regenerated leukemic cells included an initial, transient population derived from CD34+CD38+ cells as well as more sustained populations derived from CD34+CD38- progenitors, indicative of a hierarchy of transplantable leukemic cells. Analysis of the phenotypes produced revealed a reduced output of B-lineage cells, enhanced myelopoiesis with excessive production of erythroid and megakaropoietic cells and the generation of primitive (CD34+) leukemic cells displaying an autocrine IL-3 and G-CSF phenotype, all characteristics of primary CML cells. These findings demonstrate the validity of this xenograft model of chronic phase human CML, which should enable future investigation of disease pathogenesis and new approaches to therapy.     

Treatment of chronic myeloid leukemia with imatinib mesylate

Philadelphia (Ph) chromosome is the cytogenetic hallmark of chronic myeloid leukemia (CML). The translocation forms a chimeric gene, bcr-abl, which generates BCR-ABL. This fusion protein constitutively activate ABL tyrosine kinase and causes CML. Imatinib mesylate is a selective tyrosine kinase inhibitor on ABL, c-Kit and PGDF-receptor, and functions through competitive inhibition at the ATP-binding site of the enzyme, which leads to growth arrest or apoptosis in cells that express BCR-ABL. Imatinib has revolutionized the management of patients with CML, and at a dose of 400 mg daily has become the current standard therapy for newly diagnosed patients with CML even when they have HLA-matched family donors. Although imatinib therapy has only a 5-year history, it is hoped that CML will be cured with this drug and with forthcoming second-generation tyrosine kinase inhibitors as well as by allogeneic stem cell transplantation in patients who have become resistant to these drugs.     

Chronic myeloid leukaemia: an investigation into the role of Bcr-Abl-induced abnormalities in glucose transport regulation

In chronic myeloid leukaemia (CML) expression of the chimeric tyrosine kinase, Bcr-Abl, promotes the inappropriate survival of haemopoietic stem cells by a nonautocrine mechanism in the absence of IL-3. Stimulation of glucose uptake appears to play an important role in the suppression of apoptosis by this cytokine in normal haemopoietic cells. To investigate whether the cell survival mechanisms mediated by the oncoprotein and cytokine showed any similarities, we employed a haemopoietic cell line, TonB210, engineered for inducible expression of Bcr-Abl. Tyrosine kinase expression in cytokine-deprived cells was found to mimic the effect of IL-3 in maintaining a higher V(max) for hexose uptake. In both IL-3- treated cells and those expressing Bcr-Abl, high rates of hexose uptake were associated with the retention at the cell surface of approximately 80% of the total cellular content of the GLUT1 glucose transporter. In contrast, treatment of Bcr-Abl-expressing cells for 6 h with the Bcr-Abl kinase inhibitor Glivec (10 muM), in the absence of IL-3, led to internalization of approximately 90% of the cell-surface transporters and drastically decreased (4.4+/-0.9 (mean+/-s.e.m., 4)-fold) the V(max) for hexose uptake, without significant effect on the K(m) for this process or on the total cellular transporter content. These effects were not the result of any significant loss in cell viability, and preceded the onset of apoptosis caused by inhibition of Bcr-Abl. Both IL-3 treatment and expression of Bcr-Abl led to enhanced phosphorylation of Akt (protein kinase B). The stimulation of transport by IL-3 and Bcr-Abl in TonB210 cells was inhibitable by phosphatidylinositol 3-kinase inhibitors, indicating the involvement of this kinase in the signal transduction pathway. These findings suggest that inhibition of glucose transport plays an important role in the therapeutic action of Glivec, and that the signal transduction pathways involved in transport stimulation by Bcr-Abl may offer novel therapeutic targets for CML.     

Bcr-Abl has a greater intrinsic capacity than v-Abl to induce the neoplastic expansion of myeloid cells

The Bcr-Abl/p210 fusion protein plays a primary role in the pathogenesis of chronic myelogenous leukemia (CML). Abelson murine leukemia virus, which encodes v-Abl/p160, induces a pre-B cell leukemia/lymphoma in mice. It has been unclear whether the apparent specificity of these two abl oncogenes for myeloid versus lymphoid neoplasms is due to specific intrinsic properties of these Abl oncoproteins, or due to the properties of the target cells expressing them. We have recently shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder in mice resembling human CML. In this study, we compared Bcr-Abl/p210 and v-Abl/p160 in this mouse CML model. We found that early in the course of disease, both Bcr-Abl/p210 and v-Abl/p160 expanded early immature hematopoietic cells. Later Bcr-Abl/p210 selectively expanded myeloid cells while v-Abl/p160 primarily induced the rapid in vivo expansion of B lymphoblastic cells, along with a minor population of myeloid cells. In vitro, Bcr-Abl/p210 induced more growth of myeloid colonies from 5-fluorouracil treated bone marrow than v-Abl/p160. These results, obtained under equal bone marrow transduction/transplantation conditions, indicate that Bcr-Abl/p210 has a greater intrinsic capacity than v-Abl/p160 to induce the neoplastic growth of myeloid cells. In addition, we found that cultured cells expressing Bcr-Abl/p210 had more activated STAT5 than cells that expressed v-Abl/p160. This suggests that activation of STAT5 might be one part of the mechanism of abl oncogene disease specificity.     

Multilineage outgrowth of both malignant and normal hemopoietic progenitor cells from individual chronic myeloid leukemia patients in immunodeficient mice.

In this study the ability of malignant and normal progenitors in peripheral blood (PB) and bone marrow (BM) of CML patients in chronic phase to proliferate and produce mature progeny after transplantation into hereditary immunodeficient (SCID and NOD/SCID) mice was examined. Engraftment in NOD/SCID mice preconditioned by total body irradiation (TBI) alone was 10-fold higher than in SCID mice preconditioned by macrophage depletion and TBI, demonstrating that NOD/SCID mice are more suitable for engraftment of chronic phase CML cells. Low-density cells at cell doses of 10-30 x 10(6) and purified CD34+ cells at doses of approximately 0.2 x 10(6) engrafted NOD/SCID mice, with levels of 2 to 20% CD45+ cells with production of monocytes, granulocytes, erythroid cells, B-lymphocytes, CD34+ cells and variable frequencies of erythroid and myeloid colony-forming cells. As demonstrated by fluorescent in situ hybridization (FISH) analysis, purified human myeloid, B-lymphoid, erythroid and CD34+ cells from chimeric mouse BM contained Philadelphia-chromosome (Ph)-positive cells and Ph- cells in similar frequencies as primary cells from the CML patients. These results demonstrate that production of mature normal as well as malignant cells of multiple lineages were supported with similar efficiency. In contrast, all human erythroid and myeloid clonogenic cells detected in the mice were Ph-, which can be attributed to less efficient maintenance or more rapid differentiation of immature Ph+ cells in the mouse microenvironment. CML blast crisis cells also grew well in NOD/SCID mice, with 80-90% of human cells produced containing the Ph- chromosome. The availability of an in vivo assay that supports outgrowth of normal and malignant stem cells from chronic phase and blast crisis CML patients will facilitate examination of differential effects of growth factors, inhibitory cytokines and cytotoxic drugs on survival of normal and malignant stem cells in vivo and on progression of chronic phase CML towards blast crisis.     

Resistance of Philadelphia-chromosome positive leukemia towards the kinase inhibitor imatinib (STI571, Glivec): a targeted oncoprotein strikes back.

Cancer research within the last decades elucidated signaling pathways and identified genes and proteins that lead or contribute to malignant transformation of a cell. Discovery of the Bcr-Abl oncoprotein as the molecular abnormality causing chronic myeloid leukemia (CML) paved the way for the development of a targeted anticancer therapy. The substantial activity of imatinib mesylate (STI571, Glivec) in CML and Philadelphia (Ph)-chromosome positive acute lymphoblastic leukemia (Ph+ ALL) changed the therapeutic approach to Ph+ leukemia and rang the bell for a new era of anticancer treatment. However, when the phenomenon of relapse occurred despite continued imatinib treatment, we had to learn the lesson that imatinib can select for a resistant disease clone. If such a clone still depends on Bcr-Abl, it either carries a BCR-ABL point mutation that prevents binding of the drug or expresses the fusion protein at high levels. Alternatively, leukemia cells that harbor secondary genetic alterations resulting in Bcr-Abl-independent proliferation are selected for their growth advantage in the presence of imatinib. Point mutations in the BCR-ABL kinase domain prevent binding of imatinib but still allow binding of ATP, thus retaining Bcr-Abl kinase activity. Mutated BCR-ABL is frequently detected in cases of imatinib-resistant Ph+ leukemia and therefore represents the main challenge for the investigation of alternative strategies to either overcome resistance or to prevent the emergence of a resistant leukemic clone.     

Growth autonomy and lineage switching in BCR-ABL-transduced human cord blood cells depend on different functional domains of BCR-ABL.

The tyrosine kinase activity of p210BCR-ABL is essential to its leukemogenic potential, but the role of other functional domains in primary human hematopoietic cells has not been previously investigated. Here we show that infection of normal human CD34+ cord blood (CB) cells with a retroviral vector encoding p210BCR-ABL rapidly activates a factor-independent phenotype and autocrine interleukin-3/granulocyte colony-stimulating factor/erythropoietin production in the transduced cells. These changes are characteristic of primitive chronic myeloid leukemic (CML) cells and are important to the leukemogenicity of BCR-ABL-transduced murine hematopoietic stem cells. When BCR-ABL-transduced human CB cells were incubated with imatinib mesylate, an inhibitor of the p210BCR-ABL kinase, or when human CB cells were transduced with a BCR-ABL cDNA lacking the SH2 domain (p210DeltaSH2), factor independence was significantly reduced. In contrast, deletion of the SH2 domain had little impact on the p210BCR-ABL kinase-dependent promotion of erythropoietic differentiation also seen immediately following the BCR-ABL transduction of primitive human CB cells, but not in naturally occurring CML. Thus, p210BCR-ABL has distinct biological effects in primary human hematopoietic cells, which variably mimic features of human CML, and activation of these changes can show different dependencies on the integrity of the SH1 and SH2 domains of p210BCR-ABL.     

Differential effects of cladribine and gemcitabine on erythroid and granulocytic progenitors from patients with chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal neoplastic disease that originates in a pluripotent stem cell. Selection of normal progenitors by graft-purging may improve the outcome after autologous transplantation. In our methylcellulose assays, the nucleoside analogs cladribine (2-CdA) and gemcitabine (dFdC) showed more prominent inhibitory effects on CML than normal bone marrow (BM) progenitors. For dFdC, however, long-term incubations were necessary to achieve complete inhibition. Deoxycytidine kinase, the key enzyme of both 2-CdA and dFdC metabolisms, was only partially responsible for this differential sensitivity. We suggest that 2-CdA and dFdC might be helpful in purging of CML BM cells before autologous BM transplantation. Further studies on more primitive cells are warranted.     

Oleic acid is the active component in the mushroom Daedalea gibbosa inhibiting Bcr-Abl kinase autophosphorylation activity

The hallmark of chronic myeloid leukemia (CML) is the abnormal activity of p210(Bcr-Abl) kinase. Selective kinase inhibitors such as imatinib or nilotinib have been established successfully for the treatment of CML. Despite high rates of clinical response, CML patients can develop resistance to these kinase inhibitors mainly due to point mutations within the Abl kinase domain of the fusion protein. Previously, we reported that a crude extract of the mushroom Daedalea gibbosa inhibited kinase activity of Bcr-Abl kinase. Here we report on the identification of the active component of Daedalea gibbosa, oleic acid, which inhibited Bcr-Abl kinase autophosphorylation in Ba/F3 cells and exhibited anti-CML activity in a BCR/ABL-positive mouse model.     

Different target range and cytotoxic specificity of adaphostin and 17-allylamino-17-demethoxygeldanamycin in imatinib-resistant and sensitive cell lines.

Imatinib mesylate is a selective inhibitor of the oncogenic tyrosine kinase, Bcr-Abl, and is widely used as a first-line treatment for chronic myeloid leukaemia (CML). Prolonged monotherapy is frequently associated with patients becoming refractory to imatinib. Therefore, there is considerable interest in small molecule inhibitors which may be used either as replacements or as adjuncts to existing imatinib therapy. For this purpose, it is most likely that drugs which do not share imatinib's mechanism of action will be most valuable. We compared two such compounds with different modes of action, adaphostin and 17-allylamino-17-demethoxygeldanamycin (17-AAG), for their cytotoxic effect and ability to induce the downregulation of cellular proteins in a murine haemopoietic cell line transformed with human p210(Bcr-Abl), and two subclones resistant to imatinib owing to an Abl-kinase domain mutation (E255K) or amplification of the BCR-ABL gene, respectively. We found that, whereas 17-AAG selectively killed Bcr-Abl-positive cells and inhibited proteins dependent on heat-shock protein 90 for their stability (p210(Bcr-Abl) and Akt), adaphostin induced the downregulation of multiple cell-signalling proteins (p210(Bcr-Abl), Akt, Bcr, Abl and STAT5a) and was cytotoxic to both Bcr-Abl-positive and -negative cells. We suggest that both compounds may prove useful in the treatment of CML but caution that undesirable side-effects may result from the inhibition of multiple cell signalling proteins.     

Granulocyte-macrophage colony-stimulating factor modulation of the inhibitory effect of transforming growth factor-beta on normal and leukemic human hematopoietic progenitor cells.

Experiments were undertaken to investigate the molecular basis of primitive hematopoietic progenitor cell regulation in both the long-term culture system and in methylcellulose, particularly with a view to characterizing factors either able or unable to influence the behaviour of primitive leukemic cells from patients with chronic myeloid leukemia (CML). Long-term cultures of CML cells with or without irradiated normal marrow feeder layers were initiated from peripheral blood cells of CML patients with high white blood cell counts. Three weeks later the effect of exogenously added transforming growth factor-beta 1 (TGF-beta 1) on progenitor cycling status was examined. A single addition of 5 ng/ml TGF-beta 1 was able to reversibly arrest the otherwise uninterrupted turnover of primitive leukemic erythroid and granulopoietic progenitors for a period of up to 7 days both in the presence and absence of a normal adherent cell population. When TGF-beta 1 was incorporated into methylcellulose cultures, its ability to inhibit colony formation by CML progenitors showed the same differential activity on primitive cell types exhibited by normal progenitors. Dose-response curves for analogous populations of normal and leukemic cells were indistinguishable. Increasing the concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) in methylcellulose colony assays decreased the sensitivity displayed by normal clonogenic cells to TGF-beta 1 and no differences were detectable when CML cells were used in such regulator competition experiments. These findings support a general model of primitive hematopoietic cell regulation in which entry into S-phase is determined at the intracellular level by multiple convergent pathways that may deliver either positive or negative signals from activated cell surface receptors for distinct extracellular factors. The present study shows for the first time that primitive CML progenitors exposed to TGF-beta 1 in vitro can be transiently blocked in a noncycling state for several days without loss of viability and that the mechanisms responsible for the emergence and maintenance of a clonal population of CML cells in vivo do not appear to involve changes in their sensitivity to TGF-beta 1. It is thus unlikely that the heightened proliferative activity exhibited by primitive CML progenitors both in vivo and in long-term culture can be explained by an abnormality in the intracellular mechanisms normally activated by TGF-beta 1 receptor-ligand binding. We suggest that primitive CML cells are either defective in their ability to see (or activate) endogenously produced TGF-beta 1, or are defective in their responsiveness to another, undefined, regulator.